viruses Review Hepatitis B Virus DNA Integration: In Vitro Models for Investigating Viral Pathogenesis and Persistence Thomas Tu 1,2,* , Henrik Zhang 1 and Stephan Urban 3,4 1 Storr Liver Centre, Faculty of Medicine and Health, Westmead Clinical School and Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145, Australia; [email protected] 2 Centre for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia 3 Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany; [email protected] 4 German Center for Infection Research (DZIF), Heidelberg Partner Site, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany * Correspondence: [email protected] Abstract: Hepatitis B virus (HBV) is a globally-distributed pathogen and is a major cause of liver disease. HBV (or closely-related animal hepadnaviruses) can integrate into the host genome, but (unlike retroviruses) this integrated form is replication-defective. The specific role(s) of the integrated HBV DNA has been a long-standing topic of debate. Novel in vitro models of HBV infection com- bined with sensitive molecular assays now enable researchers to investigate this under-characterised phenomenon with greater ease and precision. This review covers the contributions these systems have made to understanding how HBV DNA integration induces liver cancer and facilitates viral persistence. We summarise the current findings into a working model of chronic HBV infection and discuss the clinical implications of this hypothetical framework on the upcoming therapeutic strategies used to curb HBV-associated pathogenesis. Citation: Tu, T.; Zhang, H.; Urban, S. Hepatitis B Virus DNA Integration: In Keywords: hepatitis B virus; HBV DNA integration; hepatocellular carcinoma (HCC); non-homologous Vitro Models for Investigating Viral end joining; microhomology-mediated end joining; HBV double-stranded linear DNA; viral persistence Pathogenesis and Persistence. Viruses 2021, 13, 180. https://doi.org/ 10.3390/v13020180 Academic Editors: Fabien Zoulim 1. Introduction and Duane P. Grandgenett Chronic infection with the human hepatitis B virus (HBV) is one of the major drivers Received: 29 December 2020 of liver disease and is the most common cause of liver cancer worldwide. Approximately Accepted: 21 January 2021 one third of the world’s population has been exposed to the virus, and ~257 million people Published: 26 January 2021 currently live with a chronic HBV infection [1,2]. Chronic HBV infections are usually life-long (with few exceptions) as the virus has developed several persistence mechanisms Publisher’s Note: MDPI stays neutral to escape immune surveillance, including replication via a highly-stable nuclear episomal with regard to jurisdictional claims in template (cccDNA) mimicking a mini-chromosome. Another mode of escaping elimination published maps and institutional affil- by the immune systems is the expression of sub-viral particles that dampen antiviral iations. immune responses by mechanisms not yet fully understood. 1.1. Natural History of Chronic Hepatitis B Chronic HBV infections are generally asymptomatic for long periods (up to decades) Copyright: © 2021 by the authors. as the virus replicates without triggering an antiviral immune response [3]. Decades later, Licensee MDPI, Basel, Switzerland. a sub-optimal immune response is raised against HBV-expressing hepatocytes through This article is an open access article yet-unknown mechanisms. The host response is usually inadequate to overcome viral distributed under the terms and persistence mechanisms, but instead leads to chronic inflammation, liver damage, and conditions of the Creative Commons hepatic disease. Attribution (CC BY) license (https:// Surveillance and ongoing care for chronic HBV infection is complicated by psychoso- creativecommons.org/licenses/by/ cial factors, including limited access to or distrust of the health care system, financial 4.0/). Viruses 2021, 13, 180. https://doi.org/10.3390/v13020180 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 180 2 of 16 burdens, social stigma, and systemic discrimination [4]. A poor cascade of care results: only ~10% of cases are diagnosed and only ~3% are adequately treated even in some developed countries [5,6]. Thus, HBV-related disease generally progresses unmonitored and kills ~880,000 people annually through liver cirrhosis and liver cancer [1,2]. The mechanisms by which HBV causes liver cancer are still not well understood and are under intense investigation. One of the major factors thought to be involved in hepato- carcinogenesis is the integration of the viral DNA genome into cellular chromosomes. This reportedly induces pro-oncogenic pathways through several means (previously reviewed in detail [7–9]), including cis-mediated mechanisms (e.g., HBV DNA integrations modu- lating expression of proximal cellular genes) or trans-mediated mechanisms (e.g., chronic expression of particular viral antigens). This review focuses on how in vitro models have helped in furthering our understanding of how integrated HBV DNA contributes to virus persistence and pathogenesis. 1.2. HBV Structure HBV is the prototypical member of the Hepadnaviridae family, which are small en- veloped, hepatotropic DNA viruses (~3.2 kbp) replicating via reverse transcription. The double-stranded DNA genome of HBV can take two forms (Figure1): relaxed circular DNA (rcDNA, generally present in ~90% of virions [10]) or double stranded linear DNA (dslDNA, ~10% of virions). Unlike the fully replication-competent relaxed circular form, the dslDNA-containing viral particles are replication-defective (detailed further below) but can integrate via non-homologous recombination into hepatocyte chromosomes. The HBV genome contains four overlapping open reading frames that encode for seven viral proteins, including: the HBV core antigen (HBcAg, which forms the viral capsid), e antigen (HBeAg, a secreted viral protein encoded by the core/pre-core ORF with reported tolerogenic prop- erties [11]), surface protein (HBsAg, which has three forms large, medium and small—or LHBs, MHBs, and SHBs—that envelop the virion), polymerase (HBV pol, which is essential for viral reverse transcription and replication), and the x protein (HBx, which regulates transcription of viral genes from the cccDNA episome). 1.3. HBV Replication HBV replication starts with rcDNA-containing HBV particles attaching to heparan sulphate proteoglycans on the surface of hepatocytes [12–14] (Figure2). This attachment sets up conditions for a high-affinity interaction between the preS-domain of LHBs and sodium taurocholate co-transporting polypeptide (NTCP), which acts as the functional receptor for HBV [15,16]. Binding is reportedly followed by clathrin-mediated endocytosis of the viral particle [17,18] and cytoplasmic release of the HBV nucleocapsid, which is subsequently transported to the nucleus and disassembles at the nuclear pore complex [19]. Viruses 2021, 13, 180 3 of 16 Viruses 2021, 13, x FOR PEER REVIEW 3 of 16 FigureFigure 1. The 1. The viral viral RNAs RNAs and andopen open reading reading frames frames (ORFs) (ORFs) expressed expressed from fromthe (A the) cccDNA (A) cccDNA and and (B) dslDNA forms of hepatitis B virus (HBV). The double red lines represent of the HBV DNA (B) dslDNA forms of hepatitis B virus (HBV). The double red lines represent of the HBV DNA genome, blue lines represent viral mRNAs, triangles indicate transcriptional promotors, and col- genome, blue lines represent viral mRNAs, triangles indicate transcriptional promotors, and coloured oured arrows represent viral ORFs. Nucleotide numbering of ORFs are shown as per Genbank Accessionarrows represent#AB241115. viral The ORFs. viral NucleotidemRNAs expressed numbering from of cccDNA ORFs are terminate shown as at per a polyadenylation Genbank Accession signal#AB241115. (poly A) Thelocated viral in mRNAs the Core expressed ORF. However, from cccDNA in dslDNA terminate form, atthe a canonical polyadenylation poly A signalsignal (polyis locatedA) located at the in5′ theend Core and mRNAs ORF. However, insteadin terminate dslDNA at form, a no then-canonical canonical cryptic poly A poly signal A signal. is located The at the mRNA50 end coding and mRNAs for the insteadHBV PreC/Core terminate and at a Polymera non-canonicalse are cryptic separated poly from A signal. its promoter The mRNA in the coding for dslDNAthe HBV form, PreC/Core leading andto loss Polymerase of their expression are separated (dashed from line). its promoter Figure generated in the dslDNA using form, BioRender leading to (https://biorender.com/).loss of their expression (dashed line). Figure generated using BioRender (https://biorender.com/). 1.3. HBV Replication HBV replication starts with rcDNA-containing HBV particles attaching to heparan sulphate proteoglycans on the surface of hepatocytes [12–14] (Figure 2). This attachment sets up conditions for a high-affinity interaction between the preS-domain of LHBs and sodium taurocholate co-transporting polypeptide (NTCP), which acts as the functional receptor for HBV [15,16]. Binding is reportedly followed by clathrin-mediated endocytosis of the viral particle [17,18] and cytoplasmic release of the HBV nucleocapsid, which is subsequently transported to the nucleus and disassembles at the nuclear pore